When articular cartilage is damaged as a result of osteoarthritis or injury, the tissue has limited capacity for repair and regeneration. As such, advanced stages of OA are typically characterized by extensive cartilage damage that can eventually lead to joint arthroplasty. There has been extensive research into techniques to repair damaged cartilage, including development of engineered cartilage constructs as replacement tissues, with limited success to date. In general, the goal of tissue engineering strategies is to produce a cartilage-like tissue that has a normal organization and molecular structure, and can sustain load. The optimal development of such engineered constructs requires knowledge of the composition and structure of the tissue at the macroscopic, microscopic and molecular levels through the full tissue depth. Mid-infrared (mid-IR) spectroscopy, a technique based on molecular vibrations, has previously been established as an important approach for cartilage evaluation. However, the penetration depth of mid-IR radiation is limited to ~10 microns, so that its use is restricted to surface evaluation. In contrast, near infrared spectroscopy (NIRS), a technique also based on molecular vibrations, utilizes higher frequency radiation that can penetrate tissue up to several millimeters in depth, and therefore has the potential to monitor the molecular structure of engineered tissue in vitro through the full tissue depth. Towards this goal, the current proposal seeks to develop NIRS as a modality to assess engineered cartilage during growth. This would enable engineered constructs to be modified with appropriate growth factors or mechanical input as required during development to optimize tissue structure. In concert with these studies, a further goal of this proposal is to develop strategies for cartilage tissue engineering and regeneration using mechanical input in the form of pulsed low intensity ultrasound (PLIUS) and growth factors, and to monitor the tissue changes using both NIRS and mid-IR. We will use an established model of cartilage tissue growth in a hollow fiber bioreactor for the majority of the studies. Together, these studies will demonstrate that engineered cartilage matrix can successfully be augmented, and that the changes in molecular components of the matrix can be monitored in vitro utilizing the novel application of the NIRS modality. Public Health Relevance Statement (provided by applicant): A significant impediment to advances in generating replacement tissues for damaged cartilage is the inability to assess the structure of an engineered tissue during growth. Near-infrared spectroscopic assessment could offer the ability to monitor tissue growth in vitro, and thus permit appropriate interventions to be undertaken on an ongoing basis to modify the tissue towards desired structural and compositional endpoints. Therefore, the development of this technique may play a truly central role in the exceedingly important field of tissue engineering by offering this capability.